水蛭提取液对体外培养恒河猴视网膜血管内皮细胞生长的影响

【作者】 刘聪慧；

【导师】 郑燕林；

【作者基本信息】 成都中医药大学 ， 中医眼科学， 2008， 博士

【摘要】 目的研究中药水蛭对体外培养视网膜血管内皮细胞的作用,探讨水蛭抗新生血管生成的作用及机制,为开辟新的抗新生血管生成药物提供依据,为中药新药研究和开发提供实验依据。方法1.体外培养恒河猴视网膜血管内皮细胞（RF/6A）,免疫组化进行细胞鉴定:2.选择终浓度为0mg/ml、8m/ml、16mg/ml、32mg/ml、64mg/ml、128mg/ml的水蛭提取液,分别作用于体外培养的RF/6A 24h后,观察细胞生长情况,以MTT法测定OD值,并计算出24h半数抑制浓度IC₅₀,筛选最佳抑制作用的药物浓度;3.选择终浓度为0u/ml、2.5u/ml、5u/ml、10u/ml、20u/ml的凝血酶,分别作用于体外培养的RF/6A 24h后,观察细胞生长情况,以MTT法测定OD值,筛选最佳诱导增殖的药物浓度:4.应用流式细胞术,观察水蛭提取液对体外培养的RF/6A细胞周期的影响:5.应用酶联免疫法,检测各药物组内细胞培养液中MMP-2的含量;6.应用放射免疫法,检测各药物组内细胞培养液中Ⅳ-C的含量。结果1.RF/6A具有血管内皮细胞的特点,Ⅷ因子相关抗原表达阳性,可以用于眼部病变的研究:2.体外培养的RF/6A指数增长期为48-72h;3.高浓度水蛭提取液对体外培养的视网膜血管内皮细胞具有抑制增殖作用,低浓度有促进增殖的作用:24hIC₅₀为97mg/ml;选择64mg/ml水蛭提取液为本实验的最佳抑制浓度;4.20u/ml的凝血酶对体外培养的视网膜血管内皮细胞具有促进增殖作用,选择此浓度用于后期的实验:5.水蛭提取液将体外培养中视网膜血管内皮细胞的细胞周期抑制于G1期;凝血酶组细胞多处于S期;水蛭提取液抑制了凝血酶对视网膜血管内皮细胞的作用;6.水蛭提取液可以降低体外培养视网膜血管内皮细胞的ECM中MMP-2的含量,并随时间的增加,其作用有减弱的趋势;凝血酶组随着时间的增加,其作用有增强的趋势;7.水蛭提取液可以促进体外培养视网膜血管内皮细胞的ECM中Ⅳ-C的合成,并随作用时间的持续,合成有增加趋势;凝血酶组随着作用时间的增加,有轻度促Ⅳ-C的合成增加的趋势,但与正常组相比,有降低Ⅳ-C合成,促进ECM降解作用;2h时水蛭提取液有降低凝血酶促进ECM降解的作用。结论1.64mg/ml水蛭提取液对体外培养的视网膜血管内皮细胞具有抑制作用:2.64mg/ml水蛭提取液可以改变体外培养的凝血酶诱导的视网膜血管内皮细胞的生长周期,对凝血酶诱导的细胞增殖有抑制作用:3.64mg/ml水蛭提取液促进了体外培养中凝血酶诱导下细胞微环境中Ⅳ-C的合成、抑制了MMP-2的分泌,改变了细胞生长的微环境,对新生血管的形成有抑制作用。更多还原

【Abstract】 OBJECTIVETo study the action of leech on retinal vascular epithelium cell in vitro,probe the action and mechanism of leech on anti-Vascularization,and give the prove for exploitation and study on new anti-Vascularization medicine.METHODS1.Cultured RF/6A in vitro,and identified cell by immunohistochemical method;2.Cultured RF/6A in vitro with different final concentration of Extract of leech （0mg/ml、8mg/ml、16mg/ml、32mg/ml、64mg/ml、128mg/ml）,used Colorimetry MTT to detect viability of RF/6A at 24 hour,and calculated the 50%inhibitory concentration(IC₅₀) for choosing the right concentration of Exact of leech at 24 hour;3.Cultured RF/6A in vitro with different final concentration of thrombin（0u/ml、2.5u/ml、5u/ml、10u/ml、20u/ml）,and used Colorimetry MTT to detect viability of RF/6A at 24 hour for choosing the right concentration of thrombin;4.Used flow cytometry to research the influence of Group Extract of leech、Group thrombin、Group mixing and the control on cell cycle of RF/6A cultured in vitro;5.Used euzymelinked immunosorbent assay to study the influence of every group on MMP-2 of RF/6A cultured in vitro;6.Used radio-immunity to study the influence of every group onⅣ-C of RF/6A cultured in vitro.RESULTS1.RF/6A had the feature of vascular epithelium cells,had positive antigen presentation of factorⅧ,could be usede to study the ophthalmic diseases;2.Exponential phase of growth of RF/6A cultured in vitro was the 48-72 hour;3.High concentration of Extract of leech had inhibition on proliferation of retinal vascular endothelial cell cultured in vitro,low concentration had enhancement on proliferation,the 50%inhibitory concentration(IC₅₀) at 24 hour was 97mg/ml,Extract of leech of concentration 64mg/ml was chosen to be the right concentration for this experiment;4.Thrombin of concentration 20u/ml had enhancement on proliferation of retinal vascular endothelial cell cultured in vitro,and was chosen to be the right concentration for this experiment;5.Most cells cultured in vitro were in the phrase G1 with Extract of leech,in the Group thrombin most were in the phrase S,Extract of leech had inhibition on the action of thrombin on generation cycle of retinal vascular endothelial cell cultured in vitro;6.The level of MMP-2 was decreased in Group Extract of leech,and it was increased in Group thrombin in vitro;7.The synthesis ofⅣ-C was increased in Group Extract of leech in vitro,and it was lightly increased in Group thrombin,while controlled with the normal,it was lower in Group thrombin,thrombin could enhance the degradation of ECM,which could be weekened by leech at 2h.CONCLUSION1.Extract of leech of concentration 64mg/ml has the inhibition on proliferation of retinal vascular endothelial cell cultured in vitro;2.Extract of leech of concentration 64mg/ml can change the growth cycle of retinal vascular endothelial cell cultured in vitro and inhibit the cell proliferation induced by thrombin;3.Extract of leech of concentration 64mg/ml can increase the synthesis ofⅣ-C, decrease the secretion of MMP-2 in the cell microenvironment cultured in vitro induced by thrombin,and change the microenvironment,maybe Extract of leech has the effect on anti-Vascularization.更多还原